Cellular-based Assays to Detect Toxic Agents and Elucidate the Underlying Genetic and Biochemical Mechanisms of ToxicityMichael S. Dubow, PhDMcGill University, Montreal, QuebecThe hypothesis of this research is that cells will augment or repress the expression of specific genes upon exposure to a toxic agent, in order to reorient cell physiology to cope with the stress. To identify and quantitate the activity of these genes, the investigators have prepared a library of luciferase gene fusions in the genetically well-characterized bacterium E. coli, and have identified a number of clones which illustrate luminescence changes upon cellular exposure to a variety of compounds. These clones will be validated for their use as luminescent "biosensors" to detect toxic agents, and due to the luciferase "gene tagging," the genes and encoded proteins will be isolated to identify the underlying mechanisms of cellular toxicity.Cytokine Gene Expression and Allergic Contact DermatitisCraig A. Elmets, MDCase Western Reserve University, Cleveland, OhioThe overall goal of Elmets and colleagues it to obtain a greater understanding of the role that cytokines play in allergic contact dermatitis and to use that information to develop an in vitro assay to predict the allergenicity of new products and chemicals. Using urushiol as a prototype, the investigators have found that contact allergens augment steady state levels of several epidermal cytokine genes in vivo and in vitro. They hypothesize that contact allergens can be distinguished from non-allergens by their ability to upregulate cutaneous cytokine genes in vivo and in vitro, and can be differentiated from irritant chemicals by an inherent property of the latter to upregulate HGPRT gene expression. Reverse transcriptase PCR and in situ hybridization will be performed on cultured keratinocytes and purified Langerhans cells to address these issues.Epidermal Cells Expression of Co-Stimulatory Activity in the Induction of Allergic Contact DermatitisAnthony A. Gaspari, MDUniversity of Rochester, Rochester, New YorkThe goal of this proposal is to study the regulation of antigen presenting cell functions by epidermal cells and define its relationship to allergic contact dermatitis. In the first three years of this study, Gaspari and colleagues defined the relationship between the expression of B7/BB1 antigen by epidermal cells and allergic contact dermatitis. To investigate the effects of allergens on the expression of BB-1 antigens by human keratinocytes (KC) in vitro, the investigators will analyze the effects of allergens on the regulation of the expression of cell surface B7 by cultured KC, develop a screening ELISA to study the effects of large numbers of allergens on B7 expression by cultured KC, and study the effects of allergens on cultured KC for their effects on the expression of B7 transcripts. The overall aim of the project is to better understand the immunopathologic mechanisms of allergic contact dermatitis and to use this information to develop an in vitro alternative to whole animal testing to screen for contact allergens.Standardization and Simplification of an In Vitro Assay to Screen the Efficacy of Anti-Metastatic AgentsMargaret B. Penno, PhDJohns Hopkins School of Medicine, Baltimore, MarylandThe third year grant is the final step in the development of an in vitro assay to measure tumor cell attachment to extracellular matrices. With the aid of a robotic workstation, Penno and colleagues will adapt the ceulllular attachment assay so that 96 samples (tumor cells with or without inhibitors) can be tested for attachment simultaneously. The automated method will be compared and contrasted to the manual method developed over the past two years. A cost analysis of mass drug screening using the automated system will also be performed. This study has been designed to develop a rapid and accurate in vitro test for the identification of anti-metastatic agents and provide investigators with an alternative to animal testing. The assay will also facilitate the quantitative measurements of cellular attachment involving numerous other cell adhesion molecules involved in cancer, viral infection and inflammatory disease.Intelligent Culture Dishes for Toxicity TestingNorman F. Sheppard, Jr., PhDJohns Hopkins University, Baltimore, MarylandThe aim of this project is to improve the efficacy of cytotoxicity testing by developing a system for monitoring cellular metabolism in vitro. This system will be used with mammalian cell models to test the hypothesis that knowledge of the culture medium chemistry will provide greater insight into the processes underlying cytotoxicity. The specific aims for the second year of the project are to establish a correlation between rates of consumption of metabolites such as glucose and amino acids, and/or production of metabolic products such as lactate in existing in vitro assays and to design and construct a multi-well culture plate with integral biosensors, using transparent substrate and electrode materials and associated instrumentation.In Vitro Assay for Hapten-Specific Priming of Human T LymphocytesJ. Wayne Streilein, MDSchepens Eye Research Institute, Boston, MassachusettsAn overwhelming body of data supports the contention that Langerhans cells (LC) are the predominant antigen-presenting cells in skin. Cultured Langerhans cells are believed to display properties typical of Langerhans cells that have migrated from the skin, carrying immunogenic signals to draining lymph node where the initial stages of T cell activation and sensitization begin. The investigators have demonstrated that murine Langerhans cells cultured for three days and then hapten-derivatized display the capacity to activate unprimed hapten-specific cells. Human LC cultured in vitro for three days display functional properties similar to cultured murine cells. The investigators will study the antigen-specific cellular events associated with induction and expression of human contact hypersensitivity using skin or blood as a cellular source to determine whether human epidermal and blood cells can be manipulated in vitro in order to activate naive T cells that mediate contact hypersensitivity.Development of an In Vitro Model for Human Response to DioxinThomas R. Sutter, PhDJohns Hopkins School of Public Health, Baltimore, MarylandIn this thrid year grant, Sutter and colleagues continue development of an epithelial cell culture system for measurements of human response to TCDD (dioxin). Previous studies have led to the discovery of a growth factor autocrine loop that significantly alters the cellular dose-response of human epidermal keratinocytes (HEKs) to TCDD. Specific objectives at this stage of the project include assessment of the TCDD concentration-response relationships for decreased epidermal growth factor receptor (EGF-R) binding and cellular differentiation in a single strain of HEKs and determiniation of TCDD concentration-response relationships for multiple strains of HEKs.Biochemical Characterization of ML-1 Cell Differentiation for Toxicological StudiesMichael A. Trush, PhDJohns Hopkins School of Public Health, Baltimore, MarylandThe primary objective of Trush and colleagues is to use the human myeloblastic leukemia cell line, ML-1, as a model to evaluate the effects of xenobiotics on bone marrow myeloid stem cells and on monocytic differentiation. The specific aims of this third year grant are to examine the roles of tumor necrosis factor (TNF) and transferrin in the development of mitochondrial and NADPH oxidase activity, to examine the regulation of glutathione and quinone reductase during mononuclear cell differentiation, to examine the effects of lead on ML-1 cell differentiation and to use ML-1 cells to study the effects of benzo(a)pyrene-derived quinones on mitochondrial respiration and monocyte function. The lone term objective of the work is to extend the concepts learned with this myeloid cell system to other cell differentiation models of toxicological relevance and concern.Role of Epithelium-Derived Cytokines in Oxidant-Induced InflammationMarsha Wills-Karp, PhDJohns Hopkins School of Public Health, Baltimore, MarylandWills-Karp and colleagues hypothesize that ozone-exposed epithelial cells regulate the inflammatory response to ozone by releasing cytokines which activate leukocytes. Specifically, they will determine the optimal conditions for exposing BEAS-2B cells to ozone in vitro by conducting and characterizing air and ozone exposure chambers, determine the profile of proinflammatory cytokines produced by BEAS-2B cells following ozone exposure in vitro, and determine the role of epithelial-derived cytokines in the activation of leukocytes following ozone exposure by examining the effects of culture supernatants from ozone-exposed BEAS-2B cells on leukocyte activation and cytokine production.